This proposal is designed to: 1) isolate the pig kidney 25-hydroxy vitamin D 1-hydroxylase enzyme (cytochrome P-450D1), 2) isolate cDNA for kidney ferredoxin and cytochrome P-450-D1, and 3) use isolated DNA probes to study the molecular mechanism for regulating kidney 1-hydroxylase activity. Cytochrome P-450D1 will be purified by using ligand and monoclonal affinity chromatographic procedures, and its N-terminal amino acid sequence analyzed. Using a pig kidney cDNA library which contains a high proportion of full-length copies, specific cDNA clones will be identified by colony hybridization using specific DNA probes for the two proteins. Homologous DNA mixed probes will be synthesized (14-deoxynuleotide oligomer) using the N-terminal amino acid sequence data for the P-450D1 and ferredoxin proteins. Putative cDNA clones will be verified by DNA sequence analysis in conjunction with their known N-terminal amino acid sequence. The cDNA clones will be used as molecular probes to measure changes in mRNA transcripts for kidney ferredoxin and cytochrome P-450D1 during different regulatory states. Similarly, changes in cellular enzyme activity and content will be compared using enzyme assays and radioimmunoassays (RIA). Possible enzyme-level regulation of the two enzymes activity, e.g., phosphorylation/dephosphorylation, will be investigated by using isolated kidney cells and monospecific antibodies to follow 32Pi incorporation into the proteins' during different regulatory challenges. Results and molecular probes obtained from this investigation will be used for future studies into the organization of the cytochrome P-450D1 and ferredoxin genes. It is apparent that understanding the molecular properties of the kidney-mitochondrial 1-hydroxylase enzyme system will have a profound influence on the prevention and treatment of mineral diseases. Of special interest is the long range impact this study will have on the treatment of genetic-based mineral diseases in which recombinant DNA technology could be used to treat enzyme deficiencies and abnormalities.